CN110257451A - A method of promoting sweet wormwood acid accumulation - Google Patents
A method of promoting sweet wormwood acid accumulation Download PDFInfo
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- CN110257451A CN110257451A CN201910520954.8A CN201910520954A CN110257451A CN 110257451 A CN110257451 A CN 110257451A CN 201910520954 A CN201910520954 A CN 201910520954A CN 110257451 A CN110257451 A CN 110257451A
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Abstract
The present invention provides a kind of methods for promoting sweet wormwood acid accumulation, its vegetable fat being easy to get by addition foreign aid's regulatory factor into culture medium and inexpensively, and during can produce the saccharomyces cerevisiae engineered yeast fermented and cultured of Arteannuic acid, magnesium salts and amino acid are added into culture medium, and stage regulation is carried out to fermentation temperature and fermentation dissolved oxygen, thus in ensuring saccharomyces cerevisiae engineered yeast on the basis of plasmid stability, it is greatly promoted the accumulation of Arteannuic acid, Arteannuic acid fermentation yield highest is set to can be improved 50% or more, and fermentation period also effectively shortens, significant effect.Technical solution provided by the present invention is simple and easy, further improves the production efficiency of Arteannuic acid, is easy to the industrialized production and application of Arteannuic acid.
Description
Technical field
The present invention relates to industrial microorganism fermentation technical fields, and in particular to a method of promote sweet wormwood acid accumulation.
Background technique
Qinghaosu is the sesquiterpene lactone drug for having peroxy-radical extracted from compound inflorescence plant Artemisia annua cauline leaf, point
Minor is C15H22O5, by Chinese pharmacy man slaughter cry of a deer 1971 find.Qinghaosu is after pyrimethamine, chloroquine, primary quinoline
Most effective antimalarial specific drug has the characteristics that quick-acting and less toxic, Zeng Beishi especially for encephalic malaria and anti-chlorine quinoline malaria
It is " the only effective malaria treatment drug in the world " that boundary's health organization, which is referred to as,.Its anti-malarial mechanism of action, which essentially consists in, is treating
The process of malaria is generated free radicals by qinghaosu activation, and free radical and malaria original protein binding act on the membrane system knot of plasmodium
Structure destroys its vacuolar membrane, nuclear membrane and plasma membrane, and mitochondrial swelling, interior outer membrane falls off, thus to the cell knot of plasmodium
Structure and its function damage.
In qinghaosu self-discovery so far nearly 50 years, it is widely used in related diseases fields such as treatment malaria, it is close
Year, with scientists from all over the world continue the study found that artemisinine anti-schistosome, adjusting or inhibit body fluid immune function, mention
The conversion ratio of high lymphocyte, cholagogue, eliminating the phlegm, antibechic also show that tempting prospect in the treatment for equal other diseases of relievining asthma, together
When, data are shown according to statistics, the annual whole world for qinghaosu and its derivative sales volume just up to multi-million dollar, because
This, qinghaosu market prospects are boundless.
Currently, qinghaosu can be obtained by three kinds of methods, one is traditional to extract from sweet wormwood herb and obtains, this method
Influenced by weather, region big, yield is unstable, causes qinghaosu cost of material to fluctuate year by year larger；Second is that passing through chemistry
Fully synthetic acquisition, the method difficulty is very big, and reaction process is complicated, severe reaction conditions, meanwhile, reaction cost is high；The third
Method is to obtain Arteannuic acid, then the Arteannuic acid that fermentation is obtained through changing by microbial fermentation culture by technique for gene engineering
Synthesis is learned, qinghaosu is finally obtained, which adds the method for molecular design compared with plant extract and chemistry are fully synthetic
The advantages that mild with reaction condition compared with for, yield is high, and production is stablized, easy to industrialized production.In view of above-mentioned advantage, lead to
The method for crossing microbial fermentation obtains Arteannuic acid, and the method for being then chemically synthesized production qinghaosu has been widely accepted and has obtained
To popularization and application, 2013, the qinghaosu that WHO has also approved technique production was applied to clinic.
Microbial fermentation adds molecular design to prepare in the method for qinghaosu, fermentation costs, the technological feasibility of Arteannuic acid
And the factors such as fermentation yield are that can qinghaosu efficient, low cost acquisition key influence factor.The microbial fermentation of Arteannuic acid
Method develops the support project of Gates Foundation in 2004 of starting from, both at home and abroad used by technology be develop based on this and
Come.It is existing currently, the saccharomyces cerevisiae engineered yeast for producing Arteannuic acid constructed by genetic engineering means during the fermentation, is still deposited
Fermentation costs are higher, use the poisonous and harmful substances such as a large amount of organic solvents in fermentation production process, and fermentation period is longer etc.
Problem, the cost for eventually leading to biosynthesis qinghaosu still can not contend with the qinghaosu obtained by plant extraction process
And the qinghaosu for causing biology plus molecular design method to obtain ultimately fails to form large-scale industrialization production and application.Text
Chapter High-level semi-synthetic production of the potent antimalarial artemisinin
Using in (CJ Paddon, PJ Westfall, DJ Pitera, et.al.Nature, 2013,496 (7446): 528-532) has
A large amount of additions of solvent isopropyl myristate (IPM) are unfavorable for the control of cost, and strain genetic stability is poor, yield
Unstable, fermentation period is longer, is unfavorable for industrialization large-scale production.Article Production of amorphadiene in
yeast,and its conversion to dihydroartemisinic acid,precursor to the
antimalarial agent artemisinin(PJ Westfall,DJ Pitera,JR Lenihan,
et.al.Proceedings of the National Academy of Sciences.2012,109(3):E111-E118)
It is middle to describe a kind of by way of to saccharomyces cerevisiae engineered yeast genetic modification, realize the high yield of amorpha-4,11-diene, highest
Yield is up to 40g/L, still, from amorpha-4,11-diene to Arteannuic acid, then arrives qinghaosu, centre still needs multi-step chemical
Synthesis, and yield is lower, therefore, practical industrial applications are poor.Patent CN101338309A disclose one kind pass through by
GYP71AV1 P450 gene and P450 reductase CPR channel genes brewing yeast cell come obtain Arteannuic acid genetic engineering bacterium from
And the method for producing Arteannuic acid, this method describe the process optimization and yield of Arteannuic acid fermentation yield less；Patent
CN105316372A discloses a kind of method of fermenting and producing Arteannuic acid, by largely adding n-hexane, just into fermentation liquid
The organic solvents such as heptane, toluene, dimethylbenzene or meta-xylene realize the extraction of Arteannuic acid in fermentation liquid, to improve Arteannuic acid
Yield, still, the organic reagents such as n-hexane used in the technology or normal heptane have low boiling point, volatile disadvantage, first
The organic reagents such as benzene or dimethylbenzene then have strong carcinogenicity, and therefore, real attenuation produces poor operability, and does not meet environment
The theory of friendly and sustainable development.
My company is leading to batch indirect fermentation to overcome strain Character instability present in Arteannuic acid fermentation technique
Output fluctuation is larger, in fermentation process a large amount of additions of galactolipin and organic solvent cause Arteannuic acid fermentation costs it is high,
And since the use of a large amount of organic solvents is also unfavorable for environmental protection, it is difficult to realize the disadvantages of cleaning green sustainability production,
Therefore, we have developed a kind of method for improving Arteannuic acid fermentation yield, it is specifically disclosed in CN108611383A, this method has
Strain genetic stability is high, has thoroughly abandoned the use of poisonous and harmful organic solvent, has effectively reduced fermentation by lactose hydrolysis
Cost and fermentation yield such as are obviously improved at the advantages, but the technology still have longer fermentation period, lactose hydrolysis complex process,
Still addition organic solvent, the problems such as fermentation costs are still higher in culture medium.
Summary of the invention
It is long for sweet wormwood acid fermentation zymotechnique complexity existing in the prior art, high production cost, fermentation period, and
The disadvantages of organic solvent is added in fermentation process, the present invention provides a kind of methods for promoting sweet wormwood acid accumulation.
A method of promoting sweet wormwood acid accumulation, comprising the following steps:
1) before fermentation starts, foreign aid's regulatory factor and vegetable oil are added into saccharomyces cerevisiae engineered yeast fermentation medium
Rouge；
2) during saccharomyces cerevisiae engineered yeast fermented and cultured, magnesium salts and amino acid are added into culture medium；
3) during the entire fermented and cultured of saccharomyces cerevisiae engineered yeast, stage tune is carried out to fermentation temperature and fermentation dissolved oxygen
Control.
In preferred embodiments, the foreign aid's regulatory factor added into saccharomyces cerevisiae engineered yeast fermentation medium
Addition for the one or more of isovaleric acid, isobutyric acid and 2-Methyl Butyric Acid, foreign aid's regulatory factor makes it in fermented and cultured
The concentration of base is 0.5-5g/L.
In preferred embodiments, the vegetable fat added in the saccharomyces cerevisiae engineered yeast fermentation medium is beans
The one or more of oil, corn oil, rapeseed oil and cottonseed oil, it is furthermore preferred that the addition volume of the vegetable fat is fermentation training
Support the 5-35% of base initial volume.
In preferred embodiments, the addition time of the vegetable fat is that fermentation starts preceding addition, and addition manner is
Disposable addition.
In preferred embodiments, it during the saccharomyces cerevisiae engineered yeast fermented and cultured, is added into culture medium
Magnesium salts is one or both of magnesium nitrate, magnesium sulfate, it is furthermore preferred that the addition time of the magnesium salts is the 30- of fermentation
45h, the addition of the magnesium salts make its concentration 2-10g/L in the fermentation medium.
In preferred embodiments, it during the saccharomyces cerevisiae engineered yeast fermented and cultured, is added into culture medium
Amino acid is valine, it is furthermore preferred that the addition time of the amino acid is the 30-45h of fermentation, the addition of the amino acid
Make its concentration 0.2-5g/L in the fermentation medium.
In preferred embodiments, in the fermentation process temperature regulation are as follows: fermentation start when, fermentation temperature is
It 28-32 DEG C, ferments after 40-55h, fermentation temperature is down to 22-25 DEG C, until fermentation ends.
In preferred embodiments, in the fermentation process dissolved oxygen regulation are as follows: fermentation start when, fermentation dissolved oxygen be
After 30-50%, the 40-55h that ferments, fermentation dissolved oxygen is down to 10-20%, until fermentation ends.
In preferred embodiments, the saccharomyces cerevisiae engineered yeast fermentation period is 65-75h.
In preferred embodiments, the fermentation medium of the saccharomyces cerevisiae engineered yeast are as follows: glucose 15-30g/L；Half
Lactose 2-10g/L；Ammonium sulfate 8-15g/L；Potassium dihydrogen phosphate 5-8g/L；Epsom salt 2-5.2g/L；Vitamin solution 8-
12ml/L；Metal ion solution 6-10ml/L；CuSO4·5H2O 20-40ug/L, remaining is water, with 13-14.5mol/L containing ammonia
Aqueous solution regulate and control pH to 5.0-5.5.
Wherein, the metal ion solution ingredient are as follows: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；
CoCl2·6H2O 0.47g/L；NaMoO4·2H2O 0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O 2.8g/L；
0.5M EDTA 80ml/L, remaining is water.
Wherein, the vitamin solution ingredient are as follows: biotin 0.05g/L；Calcium pantothenate 1g/L；Niacin 1g/L；Inositol 25g/
L；Vitamin B1 1g/L；Pyridoxal 1g/L；P-aminobenzoic acid 0.2g/L, remaining is water.
In preferred embodiments, further include stream plus carbon source during the fermented and cultured, the stream plus carbon source when
Between for since the 10-18h that ferment, until fermentation ends, the stream plus carbon source is in glucose solutions and ethanol water
It is one or two kinds of.
In a further preferred embodiment, the concentration of the glucose solution is 40%-70% (unit g/
L), the concentration of the ethanol water is 80%-99% (unit L/L)；The rate of the stream plus carbon source is fermentation medium
The 0.2%-2% of initial volume/per hour.
According to zymotechnique of the present invention, the stability of plasmid in saccharomyces cerevisiae engineered yeast body is significantly greatly increased, also significant contracting
Short fermentation period, and it has been obviously promoted the accumulation of Arteannuic acid, make the output increased of Arteannuic acid nearly 50% or more, and cheap plant
The use of grease while cost is reduced, also avoids the use of poisonous and harmful reagent instead of the use of organic solvent,
Zymotechnique technology of the invention is environmentally protective, is convenient for industrial applications.
The zymotechnique of Arteannuic acid is mainly reflected in compared with the advantage of the production technology of Arteannuic acid in the prior art in the present invention
The following aspects:
(1) present invention is significantly mentioned by addition foreign aid's regulatory factor isovaleric acid, isobutyric acid, 2-Methyl Butyric Acid and valine
High Arteannuic acid fermentation yield, highest can be improved 50% or more.
(2) present invention is thoroughly abandoned the addition of various organic solvents, is being significantly reduced by the use of cheap vegetable fat
While fermentation costs, the upgrading of technology has also been led, has kept entire fermentation process environmentally protective, solving technique from source may
Product and environment bring are polluted.
(3) present invention during the fermentation, by the regulation stage by stage to fermentation temperature, the dissolved oxygen that ferments, and to culture
Magnesium salts is added in base, ensure that the stability of plasmid in saccharomyces cerevisiae engineered yeast, and then ensure that the stable, high-yielding of Arteannuic acid.
(4) simultaneously, the addition of foreign aid's regulatory factor, cheap vegetable fat use and to fermentation temperature, fermentation dissolved oxygen
Deng combined regulating, also effectively shorten the fermentation period of saccharomyces cerevisiae engineered yeast, significantly improve production efficiency, and then reduce
The fermentation costs of Arteannuic acid.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below.Obviously, described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
Strain used in the present invention is the saccharomyces cerevisiae engineered yeast that can arbitrarily produce Arteannuic acid, commercially available or in laboratory voluntarily structure
It builds, such as to buy the saccharomyces cerevisiae from China typical culture collection center (Wuhan University) deposit number CCTCC AY 92011
Bacterium is starting strain, constructs mevalonate pathway (MVA) gene, ADH1 (sweet wormwood alcohol dehydrogenase base using genetic engineering means
Cause) and ALDH1 (sweet wormwood aldehyde dehydrogenase gene), it is total that overexpression tHMGR and ERG20 gene increases half terpene substances in yeast
With the accumulation of precursor farnesyl pyrophosphate FPP, and by being overexpressed saccharomyces cerevisiae MVA path gene and ADS gene, inhibit shark
Alkene synthase expression realizes a large amount of synthesis of sweet wormwood diene, further, imports foreign gene CYP71AV1 (Cytochrome P450
Enzyme gene), to realize the synthesis of Arteannuic acid, specific strain construction method can refer to document Ro D K, Paradise E M,
Ouellet M,et al.Production of the antimalarial drug precursor artemisinic
Acid in engineered yeast [J] .Nature, 2006,440 (7086): 940-943 and Paddon CJ, Westfall
PJ,Pitera DJ,et al.High-level semi-synthetic production of the potent
Antimalarial artemisinin [J] .Nature, 2013,496 (7446): the part Methods in 528-532 describes
Method detailed or notification number be that the saccharomyces cerevisiae of Arteannuic acid can be produced disclosed in the Chinese patent of CN101338309B
(Saccharomyces cerevisiae), preferably biological deposits number are the saccharomyces cerevisiae of CGMCC NO.1861.
The strain used in the embodiment of the present invention is to buy from China typical culture collection center (Wuhan University) preservation
The S. cervisiae of number CCTCC AY 92011 is starting strain, produces sweet wormwood by fermenting of constructing using genetic engineering means
The saccharomyces cerevisiae engineered yeast of acid, specific strain construction method reference literature Ro D K, Paradise E M, Ouellet M, et
al.Production of the antimalarial drug precursor artemisinic acid in
Engineered yeast [J] .Nature, 2006,440 (7086): 940-943 and Paddon CJ, Westfall PJ,
Pitera DJ,et al.High-level semi-synthetic production of the potent
Antimalarial artemisinin [J] .Nature, 2013,496 (7446): the part Methods in 528-532 describes
Method detailed.
Unless otherwise indicated, a variety of materials used in the embodiment of the present invention and reagent are all the common materials of this field kind
And reagent, it can be obtained by conventional commercial sources.
Liquid phase detection method condition used in qinghaosu bioactivity of the present invention is as follows: chromatographic column: Diamonsil C18
(250mm × 460mm, 5 μm), mobile phase: acetonitrile: 0.2% (mass volume ratio, unit g/L) phosphate aqueous solution=65:35
(v/v), gradient elution, retention time: 25min, flow velocity: 1mL/min, sample volume: 10 μ L, Detection wavelength: 212nm, column temperature:
(30±1)℃。
Seed culture medium used in the embodiment of the present invention are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO4
8g/L, MgSO4·7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/
L, succinate buffer (0.5M, pH 5.0) 100ml/L, remaining is water, PH=5.0.
The vitamin solution used in seed culture medium, Solid media for plates and fermentation medium in the embodiment of the present invention
It is as follows with metal ion solution specific formula:
Vitamin solution are as follows: biotin 0.05g/L；Calcium pantothenate 1g/L；Niacin 1g/L；Inositol 25g/L；Vitamin B1 1g/
L；Pyridoxal 1g/L；P-aminobenzoic acid 0.2g/L, remaining is water.
Metal ion solution are as follows: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；CoCl2·6H2O
0.47g/L；NaMoO4·2H2O 0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O 2.8g/L；0.5M EDTA
80ml/L, remaining is water.
Embodiment 1:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) seed liquor obtained in step (3) fermented and cultured: is seeded to fermentation training according to the inoculum concentration of 10% (V/V)
It supports in base；
The formula of fermentation medium are as follows: glucose 25g/L；Galactolipin 10g/L；Ammonium sulfate 10g/L；Potassium dihydrogen phosphate 6g/
L；Epsom salt 5g/L；Vitamin solution 10ml/L；Metal ion solution 8ml/L；CuSO4·5H2O 30ug/L, remaining is
Water.
Before fermentation starts, isovaleric acid is additionally added in fermentation medium, makes its concentration 0.5g/ in the fermentation medium
L；The volume for adding corn oil is 5% (V/V) of fermentation medium initial volume.
In fermentation process, regulates and controls pH with the aqueous solution of the 14mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-5.5
Between.
Ferment 30h when, magnesium nitrate and valine are added into fermentation medium, makes its concentration in the fermentation medium
Respectively 2g/L and 0.2g/L.Meanwhile the 40h before fermentation, fermentation temperature are set as 28 DEG C, dissolved oxygen is set as 30%, fermentation the
For 40h until fermentation ends, fermentation temperature are then set as 22 DEG C, dissolved oxygen is set as 10%.
In addition, duration stream adds 70% (unit g/L) glucose water-soluble up to fermentation ends since fermented and cultured 15h
Liquid, stream dosage hourly is the 2% of fermentation liquid initial volume, after fermented and cultured 75h, terminates fermentation, obtains containing Arteannuic acid
Fermentation liquid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 33.7g/L.
Comparative example 1:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to the inoculum concentration of 10% (V/V) and is not added
In the fermentation medium of exogenous factor；
The formula of fermentation medium are as follows: glucose 25g/L；Galactolipin 10g/L；Ammonium sulfate 10g/L；Potassium dihydrogen phosphate 6g/
L；Epsom salt 5g/L；Vitamin solution 10ml/L；Metal ion solution 8ml/L；CuSO4·5H2O 30ug/L, remaining is
Water.Before fermentation starts, isopropyl myristate 5% is added into culture medium.
In fermentation process, regulates and controls pH with the aqueous solution of the 14mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-5.5
Between.In fermentation process, fermentation temperature is set as 28 DEG C, and dissolved oxygen is set as 30%.
Up to fermentation ends since fermented and cultured 15h, duration stream adds 70% (unit g/L) glucose solution,
Stream dosage hourly is the 2% of fermentation liquid initial volume, after fermented and cultured 120h, terminates fermentation, obtains the hair containing Arteannuic acid
Zymotic fluid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 21.5g/L.
1 embodiment 1 of table and 1 Comparative result of comparative example
By comparison it is found that fermentation start before the addition of foreign aid regulatory factor, nitre in the addition and fermentation process of corn oil
The addition of sour magnesium and valine promotees along with the application in fermentation process to fermentation techniques such as the stage regulations of temperature and dissolved oxygen
So that Arteannuic acid fermentation yield is higher by 56.74% than control group, Arteannuic acid fermentation yield improve it is significant, meanwhile, fermentation period also by
120h foreshortens to 75h, and cycle time is also more significant, and then fermentation costs are effectively reduced.
Embodiment 2:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) seed liquor obtained in step (3) fermented and cultured: is seeded to fermentation training according to the inoculum concentration of 10% (V/V)
It supports in base；
Fermentation medium are as follows: glucose 15g/L；Galactolipin 2g/L；Ammonium sulfate 8g/L；Potassium dihydrogen phosphate 5g/L；Seven water sulphur
Sour magnesium 2g/L；Vitamin solution 8ml/L；Metal ion solution 6ml/L；CuSO4·5H2O 20ug/L, remaining is water.
Before fermentation starts, isovaleric acid, isobutyric acid and 2-Methyl Butyric Acid are additionally added in fermentation medium, train it in fermentation
The concentration supported in base is respectively 1g/L；Soya-bean oil, corn oil and rapeseed oil are added respectively, at the beginning of so that its addition volume is fermentation medium
10% (V/V) of initial body product.
In fermentation process, regulates and controls pH with the aqueous solution of the 14.5mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-
Between 5.5.
Ferment 45h when, magnesium nitrate and magnesium sulfate are added into fermentation medium, makes its concentration in the medium respectively be
5g/L, and addition valine, make its concentration 5g/L in the fermentation medium.Meanwhile the 55h before fermentation, fermentation temperature
It is set as 32 DEG C, dissolved oxygen is set as 50%, until fermentation ends, fermentation temperature are then set as 25 DEG C, dissolved oxygen is set fermentation 55h
It is 20%.
In addition, duration stream adds 40% (unit g/L) glucose water-soluble up to fermentation ends since fermented and cultured 10h
The ethanol water of liquid and 80% (unit L/L), stream dosage hourly is respectively the 1% of fermentation liquid initial volume, fermentation
After cultivating 70h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 45.1g/L.
Comparative example 2:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to the inoculum concentration of 10% (V/V) and is not added
In the fermentation medium of exogenous factor；
Fermentation medium are as follows: glucose 15g/L；Galactolipin 2g/L；Ammonium sulfate 8g/L；Potassium dihydrogen phosphate 5g/L；Seven water sulphur
Sour magnesium 2g/L；Vitamin solution 8ml/L；Metal ion solution 6ml/L；CuSO4·5H2O20ug/L, remaining is water.
It before fermentation starts, additionally adds in fermentation medium isopropyl myristate 30% (V/V), in fermentation process, uses
The aqueous solution of the 14mol/L containing ammonia regulates and controls pH, maintains pH in its fermentation process between 5.0-5.5.In fermentation process, fermentation temperature
Degree is set as 32 DEG C, and dissolved oxygen is set as 50%.
In addition, duration stream adds 40% (unit g/L) glucose water-soluble up to fermentation ends since fermented and cultured 10h
The ethanol water of liquid and 80% (unit g/L), stream dosage hourly is respectively the 1% of fermentation liquid initial volume, fermentation
After cultivating 100h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 26.3g/L.
2 embodiment 2 of table and 2 Comparative result of comparative example
By comparison it is found that fermentation start before the addition of foreign aid regulatory factor, magnesium in the addition and fermentation process of vegetable oil
The addition of salt and valine, along with the application in fermentation process to fermentation techniques such as the stage regulations of temperature and dissolved oxygen, fermentation
On the basis of period foreshortens to 70h by 100h, Arteannuic acid fermentation yield is higher by 71.48% than control group, Arteannuic acid fermentation yield
While improving significant, cycle time is also more significant, and then fermentation costs are effectively reduced.
Embodiment 3:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) seed liquor obtained in step (3) fermented and cultured: is seeded to fermentation training according to the inoculum concentration of 10% (V/V)
It supports in base；
Fermentation medium are as follows: glucose 30g/L；Galactolipin 5g/L；Ammonium sulfate 15g/L；Potassium dihydrogen phosphate 8g/L；Seven water sulphur
Sour magnesium 5.2g/L；Vitamin solution 12ml/L；Metal ion solution 10ml/L；CuSO4·5H2O 40ug/L, remaining is water.
Before fermentation starts, isobutyric acid is additionally added in fermentation medium, makes its concentration 5g/L in the fermentation medium；
The volume for adding cottonseed oil is 35% (V/V) of fermentation medium initial volume.
In fermentation process, regulates and controls pH with the aqueous solution of the 13mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-5.5
Between.
Ferment 35h when, magnesium sulfate is added into fermentation medium, makes its concentration 10g/L in the fermentation medium,
And addition valine, make its concentration 1g/L in the fermentation medium.Meanwhile the 45h before fermentation, fermentation temperature are set as
30 DEG C, dissolved oxygen is set as 30%, until fermentation ends, fermentation temperature are then set as 22 DEG C, dissolved oxygen is set as fermentation 45h
20%.
In addition, duration stream adds 60% (unit g/L) glucose water-soluble up to fermentation ends since fermented and cultured 18h
The ethanol water of liquid and 90% (unit L/L), stream dosage hourly is respectively the 0.5% of fermentation liquid initial volume, hair
After ferment culture 65h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 44.7g/L.
Comparative example 3:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to the inoculum concentration of 10% (V/V) and is not added
In the fermentation medium of exogenous factor；
Fermentation medium are as follows: glucose 30g/L；Galactolipin 5g/L；Ammonium sulfate 15g/L；Potassium dihydrogen phosphate 8g/L；Seven water sulphur
Sour magnesium 5.2g/L；Vitamin solution 12ml/L；Metal ion solution 10ml/L；CuSO4·5H2O 40ug/L, remaining is water.
It before fermentation starts, is additionally added in fermentation medium isopropyl palmitate 35% (V/V), in fermentation process, with containing
The aqueous solution of ammonia 13mol/L regulates and controls pH, maintains pH in its fermentation process between 5.0-5.5.In fermentation process, fermentation temperature
It is set as 30 DEG C, dissolved oxygen is set as 30%.
Up to fermentation ends since fermented and cultured 18h, duration stream add 60% (unit g/L) glucose solution and
The ethanol water of 90% (unit L/L), stream dosage hourly is respectively the 0.5% of fermentation liquid initial volume, fermentation training
After supporting 95h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 20.8g/L.
3 embodiment 3 of table and 3 Comparative result of comparative example
By comparison it is found that fermentation start before the addition of foreign aid regulatory factor, magnesium in the addition and fermentation process of vegetable oil
The addition of salt and valine, along with the application in fermentation process to fermentation techniques such as the stage regulations of temperature and dissolved oxygen, fermentation
On the basis of period foreshortens to 65h by 95h, Arteannuic acid fermentation yield is higher by 114.9% than control group, and Arteannuic acid fermentation yield mentions
While high significant, cycle time is also more significant, and then fermentation costs are effectively reduced.
Embodiment 4:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) seed liquor obtained in step (3) fermented and cultured: is seeded to fermentation training according to the inoculum concentration of 10% (V/V)
It supports in base；
Fermentation medium are as follows: glucose 20g/L；Galactolipin 3g/L；Ammonium sulfate 9g/L；Potassium dihydrogen phosphate 7g/L；Seven water sulphur
Sour magnesium 4g/L；Vitamin solution 9ml/L；Metal ion solution 7ml/L；CuSO4·5H2O25ug/L, remaining is water.
Before fermentation starts, 2-Methyl Butyric Acid is additionally added in fermentation medium, makes its concentration in the fermentation medium
3g/L；Soya-bean oil, corn oil, rapeseed oil and cottonseed oil are added respectively, and so that it is added volume is fermentation medium initial volume
7% (V/V).
In fermentation process, regulates and controls pH with the aqueous solution of the 13.5mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-
Between 5.5.
Ferment 40h when, magnesium nitrate is added into fermentation medium, makes its concentration 8g/L in the fermentation medium,
And addition valine, make its concentration 3g/L in the fermentation medium.Meanwhile the 50h before fermentation, fermentation temperature are set as
32 DEG C, dissolved oxygen is set as 50%, until fermentation ends, fermentation temperature are then set as 22 DEG C, dissolved oxygen is set as fermentation 50h
10%.
In addition, up to fermentation ends since fermented and cultured 15h, duration stream adds the ethyl alcohol of 99% (unit L/L) water-soluble
Liquid, stream dosage hourly is the 0.2% of fermentation liquid initial volume, after fermented and cultured 68h, terminates fermentation, obtains and contain Arteannuic acid
Fermentation liquid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 47.4g/L.
Comparative example 4:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to the inoculum concentration of 10% (V/V) and is not added
In the fermentation medium of foreign aid's regulatory factor；
Fermentation medium are as follows: glucose 20g/L；Galactolipin 3g/L；Ammonium sulfate 9g/L；Potassium dihydrogen phosphate 7g/L；Seven water sulphur
Sour magnesium 4g/L；Vitamin solution 9ml/L；Metal ion solution 7ml/L；CuSO4·5H2O 25ug/L, remaining is water.
It before fermentation starts, is additionally added in fermentation medium isopropyl palmitate 28% (V/V), in fermentation process, with containing
The aqueous solution of ammonia 13.5mol/L regulates and controls pH, maintains pH in its fermentation process between 5.0-5.5.In fermentation process, fermentation temperature
Degree is set as 32 DEG C, and dissolved oxygen is set as 50%.
Up to fermentation ends since fermented and cultured 15h, duration stream adds the ethanol water of 99% (unit L/L),
Stream dosage hourly is the 0.2% of fermentation liquid initial volume, after fermented and cultured 108h, terminates fermentation, obtains containing Arteannuic acid
Fermentation liquid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 29.3g/L.
4 embodiment 4 of table and 4 Comparative result of comparative example
By comparison it is found that fermentation start before the addition of foreign aid regulatory factor, magnesium in the addition and fermentation process of vegetable oil
The addition of salt and valine, along with the application in fermentation process to fermentation techniques such as the stage regulations of temperature and dissolved oxygen, fermentation
On the basis of period foreshortens to 68h by 108h, Arteannuic acid fermentation yield is higher by 61.77% than control group, Arteannuic acid fermentation yield
While improving significant, cycle time is also more significant, and then fermentation costs are effectively reduced.
Embodiment 5:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) seed liquor obtained in step (3) fermented and cultured: is seeded to fermentation training according to the inoculum concentration of 10% (V/V)
It supports in base；
Fermentation medium are as follows: glucose 18g/L；Galactolipin 6g/L；Ammonium sulfate 12g/L；Potassium dihydrogen phosphate 5g/L；Seven water sulphur
Sour magnesium 3g/L；Vitamin solution 11ml/L；Metal ion solution 9ml/L；CuSO4·5H2O 35ug/L, remaining is water.
Before fermentation starts, isobutyric acid and 2-Methyl Butyric Acid are additionally added in fermentation medium, makes it in the fermentation medium
Concentration be respectively 2.5g/L；Rapeseed oil is added, addition volume is 30% (V/V) of fermentation medium initial volume.
In fermentation process, regulates and controls pH with the aqueous solution of the 14mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-5.5
Between.
Ferment 38h when, magnesium nitrate and magnesium sulfate are added into fermentation medium, makes its concentration in the fermentation medium
Respectively it is 4g/L, and addition valine, makes its concentration 4g/L in the fermentation medium.Meanwhile the 55h before fermentation, fermentation
Temperature is set as 28 DEG C, and dissolved oxygen is set as 40%, and fermentation 55h is then set as 23 DEG C up to fermentation ends, fermentation temperature, dissolved oxygen
It is set as 20%.
In addition, duration stream adds 70% (unit g/L) glucose water-soluble up to fermentation ends since fermented and cultured 14h
The ethanol water of liquid and 80% (unit L/L), stream dosage hourly is respectively the 0.8% of fermentation liquid initial volume, hair
After ferment culture 72h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 43.5g/L.
Comparative example 5:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to the inoculum concentration of 10% (V/V) and is not added
In the fermentation medium of foreign aid's regulatory factor；
Fermentation medium are as follows: glucose 18g/L；Galactolipin 6g/L；Ammonium sulfate 12g/L；Potassium dihydrogen phosphate 5g/L；Seven water sulphur
Sour magnesium 3g/L；Vitamin solution 11ml/L；Metal ion solution 9ml/L；CuSO4·5H2O35ug/L, remaining is water.
It before fermentation starts, is additionally added in fermentation medium dodecanoic acid 30% (V/V), in fermentation process, with containing ammonia
The aqueous solution of 14mol/L regulates and controls pH, maintains pH in its fermentation process between 5.0-5.5.In fermentation process, fermentation temperature is set
It is set to 28 DEG C, dissolved oxygen is set as 40%.
Up to fermentation ends since fermented and cultured 14h, duration stream add 70% (unit g/L) glucose solution and
The ethanol water of 80% (unit L/L), stream dosage hourly is respectively the 0.8% of fermentation liquid initial volume, fermentation training
After supporting 100h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 28.2g/L.
5 embodiment 5 of table and 5 Comparative result of comparative example
By comparison it is found that fermentation start before the addition of foreign aid regulatory factor, magnesium in the addition and fermentation process of vegetable oil
The addition of salt and valine, along with the application in fermentation process to fermentation techniques such as the stage regulations of temperature and dissolved oxygen, fermentation
On the basis of period foreshortens to 72h by 100h, Arteannuic acid fermentation yield is higher by 54.25% than control group, Arteannuic acid fermentation yield
While improving significant, cycle time is also more significant, and then fermentation costs are effectively reduced.
Embodiment 6:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) seed liquor obtained in step (3) fermented and cultured: is seeded to fermentation training according to the inoculum concentration of 10% (V/V)
It supports in base；
Fermentation medium are as follows: glucose 28g/L；Galactolipin 8g/L；Ammonium sulfate 13g/L；Potassium dihydrogen phosphate 6g/L；Seven water sulphur
Sour magnesium 2.5g/L；Vitamin solution 10ml/L；Metal ion solution 10ml/L；CuSO4·5H2O 40ug/L, remaining is water.
Before fermentation starts, isovaleric acid and 2-Methyl Butyric Acid are additionally added in fermentation medium, makes it in the fermentation medium
Concentration be respectively 1.5g/L；Soya-bean oil is added, addition volume is 25% (V/V) of fermentation medium initial volume.
In fermentation process, regulates and controls pH with the aqueous solution of the 13.5mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-
Between 5.5.
Ferment 40h when, magnesium sulfate is added into fermentation medium, makes it in the concentration 8g/L of fermentation medium, with
And addition valine, make its concentration 3.5g/L in the fermentation medium.Meanwhile the 48h before fermentation, fermentation temperature are set as
29 DEG C, dissolved oxygen is set as 40%, until fermentation ends, fermentation temperature are then set as 24 DEG C, dissolved oxygen is set as fermentation 48h
10%.
In addition, duration stream adds 50% (unit g/L) glucose water-soluble up to fermentation ends since fermented and cultured 16h
The ethanol water of liquid and 95% (unit L/L), stream dosage hourly is respectively the 1% of fermentation liquid initial volume, fermentation
After cultivating 74h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 46.6g/L.
Comparative example 6:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) bacteria selection: is diluted 10-7After times, it is solid that 100 μ L of absorption are applied to plate
On body culture medium, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from Solid media for plates, and be seeded in seed culture medium, culture is for 24 hours
Obtain seed liquor；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to the inoculum concentration of 10% (V/V) and is not added
In the fermentation medium of foreign aid's regulatory factor；
Fermentation medium are as follows: glucose 28g/L；Galactolipin 8g/L；Ammonium sulfate 13g/L；Potassium dihydrogen phosphate 6g/L；Seven water sulphur
Sour magnesium 2.5g/L；Vitamin solution 10ml/L；Metal ion solution 10ml/L；CuSO4·5H2O 40ug/L, remaining is water.
It before fermentation starts, is additionally added in fermentation medium dodecanoic acid 25% (V/V), in fermentation process, with containing ammonia
The aqueous solution of 13.5mol/L regulates and controls pH, maintains pH in its fermentation process between 5.0-5.5.In fermentation process, fermentation temperature
It is set as 29 DEG C, dissolved oxygen is set as 40%.
Up to fermentation ends since fermented and cultured 16h, duration stream add 50% (unit g/L) glucose solution and
The ethanol water of 95% (unit L/L), stream dosage hourly is respectively the 1% of fermentation liquid initial volume, fermented and cultured
After 96h, terminate fermentation, obtains the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 22.8g/L.
Comparative example 7:
(1) activation of strain: the saccharomyces cerevisiae engineered yeast strain that -80 DEG C freeze is inoculated by inoculum concentration 0.5% (V/V)
In seed culture medium, 16h is cultivated, strain liquid is obtained；
(2) strain liquid obtained in step (1) resistance screening of strain: is diluted 10-7After times, 100 μ L of absorption, which are applied to, to be contained
On the Solid media for plates for there are 200 μ L hygromycin Bs, 40h is cultivated, single colonie is obtained；
Solid media for plates formula are as follows: glucose 19.5g/L, (NH4)2SO415g/L, KH2PO48g/L, MgSO4·
7H2O 6.2g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O 40ug/L, succinic acid buffer
Liquid 100ml/L (0.5M, pH 5.0), agar 20g/L, remaining is water.
(3) seed culture: the picking single colonie from the Solid media for plates containing antibiotic, and it is seeded to seed culture
In base, culture obtains seed liquor for 24 hours；
(4) fermented and cultured: seed liquor obtained in step (3) is seeded to according to 10% inoculum concentration containing mevalonic acid
It is cultivated in the fermentation medium of citral, wherein the concentration of mevalonic acid and citral in the medium is respectively 0.1g/L；
The formula of fermentation medium are as follows: lactose hydrolysis liquid (takes 1200g lactose to be dissolved in 2L water, 12g solution is in galactoside
Enzyme is 4.0,53 DEG C of hydrolysis 4h in pH, and all lactose hydrolysis liquid is mixed into fermentation medium, and constant volume by percent hydrolysis 93%
To 20L, making the mass volume ratio of glucose and galactolipin and fermentation medium in lactose hydrolysis liquid is respectively 28:1 (g/L))；Sulphur
Sour ammonium 10g/L；Potassium dihydrogen phosphate 6g/L；Epsom salt 5g/L；Vitamin solution 10ml/L；Metal ion solution 8ml/L；
CuSO4·5H2O 30ug/L, remaining is water.
In fermentation process, regulates and controls pH with the aqueous solution of the 14mol/L containing ammonia, pH in its fermentation process is made to maintain 5.0-5.5
Between.In fermentation process, fermentation temperature is set as 29 DEG C, and dissolved oxygen is set as 40%.
Up to fermentation ends since fermented and cultured 15h, duration stream adds 70% (unit g/L) aqueous sucrose solution, often
The stream dosage of hour is the 2% of fermentation liquid initial volume, and up to fermentation ends since fermented and cultured 40h, duration stream adds
30% (unit g/L) sodium glutamate aqueous solution, stream dosage hourly is the 0.2% of fermentation liquid initial volume, fermented and cultured
After 52h, isopropyl palmitate and oleic acid are disposably added, additional amount is the 15% of fermentation liquid initial volume；Fermented and cultured 74h
Afterwards, terminate fermentation, obtain the fermentation liquid containing Arteannuic acid.
(5) bioactivity: containing the fermentation liquid of Arteannuic acid with 9.5ml methanol extraction 0.5ml, after ultrasonic vibration 30min, mistake
Filter, through HPLC detect Arteannuic acid fermentation yield be 29.6g/L.
6 embodiment 6 of table and 6 Comparative result of comparative example
By embodiment 6 and comparative example 6 it is found that the addition, vegetable oil for starting preceding foreign aid's regulatory factor of fermenting add
Add and fermentation process in magnesium salts and valine addition, along in fermentation process to hairs such as stage of temperature and dissolved oxygen regulations
The application of ferment technology, on the basis of fermentation period foreshortens to 74h by 96h, Arteannuic acid fermentation yield is higher by than control group
104.38%, while Arteannuic acid fermentation yield improves significant, cycle time is also more significant, and then fermentation costs obtain effectively
It reduces.
Comparative example 7 be CN108611383A described in method embodiment, by with 6 phase of embodiment in the present invention
Than, under the premise of identical fermentation time 74h, the sweet wormwood acid yield that method of the present invention finally obtains is 46.6g/L, and
Method described in CN108611383A is under identical fermentation condition, final sweet wormwood acid yield only 29.6g/L, therefore, the present invention
Patent promotes Arteannuic acid potency obvious.
Claims (12)
1. a kind of method for promoting sweet wormwood acid accumulation, it is characterised in that: 1) before fermentation starts, ferment to saccharomyces cerevisiae engineered yeast
Foreign aid's regulatory factor and vegetable fat are added in culture medium；2) during saccharomyces cerevisiae engineered yeast fermented and cultured, to culture medium
Middle addition magnesium salts and amino acid；3) molten to fermentation temperature and fermentation during the entire fermented and cultured of saccharomyces cerevisiae engineered yeast
Oxygen carries out stage regulation.
2. a kind of method for promoting sweet wormwood acid accumulation as described in claim 1, it is characterised in that the saccharomyces cerevisiae engineered yeast
The foreign aid's regulatory factor added in fermentation medium is the one or more of isovaleric acid, isobutyric acid and 2-Methyl Butyric Acid, described outer
It helps regulatory factor and adds the concentration 0.5-5g/L for making it in the fermentation medium.
3. a kind of method for promoting sweet wormwood acid accumulation as described in claim 1, which is characterized in that the saccharomyces cerevisiae engineered yeast
The vegetable fat added in fermentation medium is the one or more of soya-bean oil, corn oil, rapeseed oil and cottonseed oil, the vegetable oil
The addition volume of rouge is the 5-35% of fermentation medium initial volume.
4. a kind of method for promoting sweet wormwood acid accumulation as described in claim 1, which is characterized in that the addition of the vegetable fat
Time is that fermentation starts preceding addition, and addition manner is disposable addition.
5. a kind of method for promoting sweet wormwood acid accumulation as described in claim 1, which is characterized in that the saccharomyces cerevisiae engineered yeast
During fermented and cultured, the magnesium salts added into culture medium is the one or two of magnesium nitrate and magnesium sulfate, and the magnesium salts adds
It is the 30-45h of fermentation between added-time, the addition of the magnesium salts makes its concentration 2-10g/L in the fermentation medium；The wine
In brewer yeast engineering bacterium fermentation incubation, the amino acid that adds into culture medium is valine, when the addition of the amino acid
Between for fermentation 30-45h, the addition of the amino acid makes its concentration 0.2-5g/L in the fermentation medium.
6. a kind of method for promoting sweet wormwood acid accumulation as described in claim 1, which is characterized in that the saccharomyces cerevisiae engineered yeast
The regulation of temperature in fermentation process are as follows: when fermentation starts, fermentation temperature is 28-32 DEG C, after the 40-55h that ferments, fermentation temperature drop
To 22-25 DEG C, until fermentation ends；The regulation of dissolved oxygen in the saccharomyces cerevisiae engineered yeast fermentation process are as follows: when fermentation starts, hair
Ferment dissolved oxygen is 30-50%, and after the 40-55h that ferments, fermentation dissolved oxygen is down to 10-20%, until fermentation ends.
7. a kind of method for promoting sweet wormwood acid accumulation as claimed in any one of claims 1 to 6, which is characterized in that the wine brewing ferment
Female engineering bacterium fermentation cultivation cycle is 65-75h.
8. such as a kind of described in any item methods for promoting sweet wormwood acid accumulation of claim 1-7, which is characterized in that the wine brewing ferment
The fermentation medium of female engineering bacteria are as follows: glucose 15-30g/L；Galactolipin 2-10g/L；Ammonium sulfate 8-15g/L；Potassium dihydrogen phosphate
5-8g/L；Epsom salt 2-5.2g/L；Vitamin solution 8-12ml/L；Metal ion solution 6-10ml/L；CuSO4·5H2O
20-40ug/L, remaining is water, regulates and controls pH to 5.0-5.5 with the aqueous solution of the 13-14.5mol/L containing ammonia.
9. a kind of method for promoting sweet wormwood acid accumulation as claimed in claim 8, which is characterized in that the metal ion solution at
It is divided into: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；CoCl2·6H2O 0.47g/L；NaMoO4·2H2O
0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O 2.8g/L；0.5M EDTA80ml/L, remaining is water.
10. a kind of method for promoting sweet wormwood acid accumulation as claimed in claim 8, which is characterized in that the vitamin solution at
It is divided into: biotin 0.05g/L；Calcium pantothenate 1g/L；Niacin 1g/L；Inositol 25g/L；Vitamin B1 1g/L；Pyridoxal 1g/L；It is right
Aminobenzoic acid 0.2g/L, remaining is water.
11. such as a kind of described in any item methods for promoting sweet wormwood acid accumulation of claim 1-10, which is characterized in that the fermentation
It further include stream plus carbon source in incubation, the stream adds the time of carbon source to be since the 10-18h that ferments, until fermentation ends, institute
It states stream plus carbon source is one or both of glucose solution and ethanol water.
12. a kind of method for promoting sweet wormwood acid accumulation as claimed in claim 11, which is characterized in that the glucose solution
Concentration be 40%-70% (unit g/L), the concentration of the ethanol water is 80%-99% (unit L/L)；It is described
Stream plus carbon source rate be fermentation medium initial volume 0.2%-2%/per hour.
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| CN113444654A (en) * | 2019-11-06 | 2021-09-28 | 天津大学 | Saccharomyces cerevisiae engineering bacterium for producing dihydroartemisinic acid and construction method and application thereof |
| CN115386588A (en) * | 2021-05-19 | 2022-11-25 | 中国科学院分子植物科学卓越创新中心 | Method for synthesizing insect sex pheromone by yeast |
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| CN115386588A (en) * | 2021-05-19 | 2022-11-25 | 中国科学院分子植物科学卓越创新中心 | Method for synthesizing insect sex pheromone by yeast |
| CN115386588B (en) * | 2021-05-19 | 2024-04-26 | 中国科学院分子植物科学卓越创新中心 | Method for synthesizing insect sex pheromone by using yeast |
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